PROJECT SUMMARY/ABSTRACT It is now well established that the onset of T cell development is initiated by the induction of Notch expression. During the previous grant cycle, we found that this critical step is controlled by the combined activities of E2A and HEB. Once Notch1 expression is activated, Notch signaling acts in concert with RUNX1, TCF1, E2A and GATA-3 to activate Bcl11b expression. Bcl11b next acts in concert with E2A to activate a T-lineage specific gene program. We (with Project 1) also demonstrated that E2A activates the expression of a non-coding transcript, named ThymoD, to reposition the Bcl11b enhancer from the nuclear lamina to the nuclear interior. These studies linked E-protein occupancy, non-coding transcription and Bcl11b expression into a common pathway that orchestrates the ?? versus ?? T cell fate decision. We now aim to describe, in physical and kinetic terms, Bcl11b locus movement relative to the onset of T cell development. To track genomic interactions in live lymphoid cells, we have developed a novel strategy. Specifically, we tracked the motion of DNA elements to visualize VDJ recombination in live B cells using tandem arrays of WT-TET and MUT-TET repressor binding sites. We propose to use the same strategy to describe in live T cells the trajectories of Bcl11b enhancer- promoter communication. Specifically, we will examine how Bcl11b enhancer-promoter communication is suppressed when sequestered at the nuclear lamina prior to commitment to the T cell lineage and after these loci return to the lamina upon adoption of the ?? T cell fate (with Projects 1 and 3). We will also examine how E-proteins and Notch signaling modulate Bcl11b enhancer-promoter communication to establish and maintain ?? T cell identity (with Projects 2 and 3) and how E-proteins, Notch signaling, non-coding transcription and nuclear repositioning are linked in four-dimensional space to orchestrate ?? T cell fate. Collectively these studies proposed here would be a first step towards describing the ?? versus ?? T cell fate choice in four- dimensional space and would not be possible outside of this integrated programmatic effort.